The movie begins before the end of the events in The Bourne Supremacy, as Bourne, wounded by a gunshot from the Russian assassin Kirill, is still evading the Moscow police. Cornered by two officers while breaking into a medical clinic to treat his wounds, Bourne overpowers the officers, knocking one out, while holding the second at gunpoint. He leaves them alive as he escapes, saying, My argument is not with you.Six weeks later, Simon Ross, a security correspondent for The Guardian, meets with Neal Daniels, the Madrid CIA station chief, in Turin, to discuss Treadstone. Bourne goes to Paris to tell Marie's brother, Martin, of her death, then heads to London. Bourne then reads an article in The Guardian by Ross about Bourne, Treadstone, and "Operation Blackbriar." He then arranges to meet Ross in London at the south entrance of Waterloo Station. Ross, however, is under surveillance because his use of the word "Blackbriar" in a phone call to his editor was tracked by ECHELON, alerting the CIA. CIA section chief Noah Vosen alerts his staff at the Anti-Terrorism Deep Cover in New York to find out any information on Ross, believing that Operation Blackbriar has been compromised.

After receiving a call from Bourne, Ross takes a taxi to Waterloo Station. At the station, Bourne sees CIA officers following Ross and places a prepaid mobile phone on him; through it, Bourne instructs him on how to dodge the station's surveillance. However, Vosen orders an assassin, Paz, to kill Ross and his source. Vosen's team identifies Bourne on a security camera and recognizes him as the original Treadstone assassin, and assumes he is Ross's source. While Paz gets into position with a sniper rifle, Bourne tells Ross to remain hidden, but Ross panics and steps out into the open, allowing Paz to deliver a clear kill shot. In the chaos, Bourne grabs Ross's notes, revealing his source as Daniels.

Deputy Director Pamela Landy is asked to help capture Bourne. With Landy's help, Vosen and his team determine that Daniels is Ross' source. They send a team to Daniels' office in Madrid, but Bourne arrives first, finding one photograph in an otherwise empty safe as Daniels has already left. Bourne attacks the CIA team members when they enter the office. Right after Bourne secures the room, Nicky Parsons then enters it unexpectedly. Nicky tells him that she was reassigned to Madrid after Berlin, and that Daniels has fled to Tangier. She helps him escape the CIA reinforcements by telling Vosen that Bourne had already left the office. Her motivation for helping Bourne isn't entirely clear, but it is subtly implied that before he lost his memory, they had been in a relationship. While in Tangier, they realize the CIA has sent assassin Desh Bouksani to eliminate Daniels. Nicky uses her official clearance access to send Desh a message, telling him to meet her for a new phone, thus allowing Bourne to follow Desh to his target. When Vosen realizes Nicky's deceit he orders Desh to kill Nicky and Bourne after terminating Daniels. Landy is outraged at Vosen's willingness to indiscriminately kill CIA personnel and quits the operation.

Bourne follows Desh and is unable to save Daniels, who dies from Desh's planted bomb. When Desh returns for Nicky, Bourne outruns the Tangier police and attacks Desh. After a brief, intense fight, Bourne eventually strangles him. Bourne has Nicky code in the fact that the both of them are dead to give each of them a head start at disappearing. Bourne then puts Nicky on a bus with the advice that, "It gets easier." (Referring to blending in and disappearing). At the Tangier morgue, Bourne examines Daniels' charred papers and finds the address of the CIA substation in New York City. Bourne takes a flight to New York City, and on arrival deliberately uses a passport that alerts Landy to his presence. Bourne calls Landy while observing her and Vosen from across the street, the same call that was shown at the end of The Bourne Supremacy. Bourne sends a text message to Landy to arrange a meeting. Vosen and his team intercept the message and follow Landy as she leaves the building. However, the meeting is simply a diversion to allow him to enter Vosen's office and steal classified Blackbriar documents.

Vosen recognizes the diversion too late after receiving a phone call from Bourne to get a recording of his voice, which is one of the keys to unlocking his Blackbriar safe. Bourne reveals he is in Vosen's office, and Vosen frantically orders CIA officers back to his office to capture Bourne. But Bourne escapes and enters into a car chase with CIA officers and Paz. Just outside the Treadstone facility, Bourne meets Landy and gives her the stolen documents from Vosen's office before entering the building. On his way back to his office, Vosen eventually works out that Landy gave Bourne an address in code when Landy gave Bourne his date of birth. Vosen races to the facility where Landy finishes faxing away the Blackbriar document that Bourne stole to the FBI. She looks at Vosen and says, "You better get yourself a good lawyer." Inside, Bourne meets with Dr. Albert Hirsch, who ran Treadstone's psychological conditioning program. With his help, Bourne remembers that, as Captain David Webb, he volunteered for the program and killed a man in the same room. Horrified by the memory of what he did to complete his conditioning and to assume the Jason Bourne identity, he tells Hirsch, "I remember everything. I'm no longer Jason Bourne."

Breaking off from the flashback, Bourne jumps out a window when CIA agents barge into the room. After a relatively short chase to the roof Paz holds Bourne at gunpoint and demands to know why Bourne spared his life. "Do you even know why you're supposed to kill me?....Look at us. Look at what they make you give," says Bourne, repeating the dying words of the Treadstone assassin called The Professor from The Bourne Identity. Paz lowers his weapon, but Vosen appears and fires at Bourne just as he jumps from the roof into the East River below.

Some time later, Nicky sees a news report noting the exposure of Blackbriar, the arrests of Hirsch and Vosen, that Ezra Kramer is the subject of a United States Senate hearing regarding his conduct, and that David Webb was shot and fell into the East River but his body was not recovered, even after a three-day search, at which Nicky smiles knowingly. In the final moments of the film Bourne is shown swimming away in the East River.